KR20230045931A - Saccharomyces pastorianus hy2 with excellent isoamyl acetate producing ability - Google Patents

Abstract

The present invention relates to Saccharomyces pastorianus HY2 (Accession number KFCC11896P). In addition, the present invention also relates to a composition comprising the Saccharomyces pastorianus HY2, a culture thereof, a concentrate of the culture or a diluted culture.

Description

Saccharomyces Pastorianus HY2 with excellent isoamyl acetate producing ability {SACCHAROMYCES Pastorianus HY2 WITH EXCELLENT ISOAMYL ACETATE PRODUCING ABILITY}

The present invention relates to a composition comprising Saccharomyces pastorianus HY2 having an excellent ability to produce isoamyl acetate, a culture thereof, a concentrate of the culture, or a dilution of the culture.

Recently, consumer interest in the diversity of beer products is explosively increasing. For example, Korean Patent Publication No. 10-2014-0096011 discloses a method for producing beer using desalted lava seawater, and Korean Patent Publication No. 10-2014-0011396 discloses a beer-taste beverage containing saponin. In addition, there is an increasing need to develop excellent beer with enhanced flavor in addition to the characteristics of lager beer, which has a clean, cool and refreshing feeling instead of less aroma and deep taste.

Brewer's yeast undergoes a fermentation process using raw materials such as sugar and amino acids, and produces various by-products such as alcohol, aroma, and organic acids, which complete beer with a unique flavor depending on the type of brewer's yeast, fermentation conditions, and raw material quality. do.

One of the important factors influencing the quality of beer is aroma, and although the concentration of aroma production varies depending on fermentation conditions, most of the aroma components are determined by the type of yeast. Isoamyl acetate, a major aroma compound, is a representative flavor component that imparts a banana flavor to beer and is one of the important factors that can differentiate lager beer according to its concentration.

In order to secure strains with excellent fragrance production ability, various methods such as screening naturally derived strains, genetic recombination, and improvement of the beer manufacturing process have been tried. There are difficulties in application such as stability problems caused by GMOs and resistance to GMOs, and there is a limit to increasing the fragrance component through improvement of the beer manufacturing process.

Saccharomyces pastorianus, a beer lager yeast, is known to be a cross between Saccharomyces cerevisiae and Saccharomyces eubayanus, and expresses recessive genetic traits Due to this difficult characteristic, there are few cases where strain improvement methods through EMS mutagen treatment have been applied.

An object of the present invention is to provide yeast with excellent ability to produce isoamyl acetate that imparts a banana flavor to alcoholic beverages, particularly beer.

Another object of the present invention is to provide a liquor with enhanced banana flavor by using yeast having excellent isoamyl acetate producing ability.

In order to achieve the above object, the present invention,

Saccharomyces pastorianus HY2 (accession number KFCC11896P) is provided.

Also, the present invention

A composition comprising Saccharomyces pastorianus HY2, a culture thereof, a concentrate of the culture, or a dilution of the culture is provided.

Also, the present invention

Adding Saccharomyces pastorianus HY2 to the wort; and

It provides a method for producing a beverage comprising the step of fermenting the wort to which the Saccharomyces pastorianus HY2 is added.

In addition, the present invention relates to a method for producing a beverage comprising the step of adding and fermenting Saccharomyces pastorianus HY2.

The Saccharomyces pastorianus HY2 strain of the present invention is an improved strain of Saccharomyces pastorianus resistant to TFL, and has similar fermentation characteristics to the parent strain through a beer fermentation test, and isoamyl acetate component It was confirmed that it has the characteristics of biosynthesizing at high concentrations. In addition, a beverage prepared using Saccharomyces pastorianus HY2 of the present invention, in particular, has a banana flavor.

Figure 1 shows the biosynthetic pathway of isoamyl acetate (Ogata & Miyashita, ASBC, 2014)
2 is a chemical formula of DL-Leucine and 5,5,5-Trifluoro-DL-Leucine, and the chemical structures of both compounds can be compared.
Figure 3 shows the survival rate of the parent strain according to the 5 v / v% EMS treatment time.
Figure 4 shows the results of culturing the strain suspension in a TFL selection medium. Control: EMS untreated strain, Trial: EMS treated strain.

the present invention

Saccharomyces pastorianus HY2 (accession number KFCC11896P).

Also, the present invention

To a composition comprising Saccharomyces pastorianus HY2, a culture thereof, a concentrate of the culture or a dilution of the culture.

Also, the present invention

Adding Saccharomyces pastorianus HY2 of claim 1 to wort; and

It relates to a method for producing a beverage comprising the step of fermenting the wort to which the Saccharomyces pastorianus HY2 is added.

Hereinafter, the present invention will be described in detail.

Saccharomyces Pastorianus HY2

Saccharomyces Pastorianus HY2 of the present invention is Saccharomyces Pastorianus HY2 deposited under accession number KFCC11896P. Saccharomyces Pastorianus HY2 is characterized by a superior ability to produce isoamyl acetate than wild-type Saccharomyces Pastorianus.

Isoamyl acetate is one of the main flavor components of beer and is a major by-product of ethanol fermentation. Isoamyl acetate is produced from isoamyl alcohol and imparts a banana flavor to the fermented product. Melting point is about -78 ℃, solubility in water is 0.3% (20 ℃). The threshold in beer is 1.1 mg/L or more, so that consumers can feel the banana flavor in beer only when the concentration is higher than the above concentration. The chemical structure of isoamyl acetate is shown in Formula 1 below.

<Formula 1>

In addition, Saccharomyces pastorianus HY2 of the present invention has wild-type Saccharomyces pastorianus, which is the parent strain, and fermentation properties (eg, alcohol, ie, ethanol producing ability, fermentation power, final VDK concentration, yeast sedimentation, recovered yeast dead cells, etc. ) are similar, there is no significant difference in fermentation characteristics between Saccharomyces pastorianus HY2 and the parent strain.

Feedback inhibition, which effectively maintains life activities by inhibiting the production of certain compounds in excess, occurs. Brewer's yeast is also a living organism with a feedback inhibition mechanism, and in the course of the isoamyl acetate synthesis pathway, α-isopropyl-malate synthase (hereinafter referred to as α-IPM synthase) is inhibited by leucine concentration, so leucine synthesis is regulated , in the end, it is impossible to synthesize isoamyl acetate above a certain level. Therefore, in order to develop strains with excellent isoamyl acetate production ability, strains resistant to 5,5,5-Trifluoro-DL-Leucine (5,5,5-trifluoro-DL-leucine) (hereinafter referred to as TFL) should be selected, and the selected strain may release isoamyl acetate by releasing the feedback inhibition mechanism by leucine. (Tomoo Ogata and Seiko Miyashita, J. Am. Soc. Brew. Chem. 72(2):110-115, 2014) (FIG. 1).

Saccharomyces pastorianus HY2 of the present invention is a strain prepared through strain improvement without inserting a foreign gene, and has high productivity of isoamyl acetate imparting a banana flavor. That is, Saccharomyces pastorianus HY2 of the present invention produces a larger amount of isoamyl acetate than the parent strain, wild-type Saccharomyces pastorianus, when fermented under the same conditions and for the same time. Therefore, using Saccharomyces pastorianus HY2 of the present invention, banana-flavored beverages, particularly beer and low-malt beer, can be prepared. In addition, Saccharomyces pastorianus HY2 of the present invention has no significant difference in fermentation characteristics from the parent strain, wild-type Saccharomyces pastorianus, and can be used industrially because of fermentation stability.

A composition comprising Saccharomyces pastorianus HY2, a culture thereof, a concentrate of the culture or a dilution of the culture

The present invention relates to a composition comprising Saccharomyces pastorianus HY2 of the present invention, a culture thereof, a concentrate of the culture, or a dilution of the culture. The culture, concentrate and/or dilution can be consumed as food.

The composition may be a food composition. The food composition of the present invention, Saccharomyces pastorianus HY2 of the present invention, its culture, the concentrate of the culture or the dilution of the culture may be added as is or used together with other foods or food compositions, It can be used appropriately according to a conventional method. There is no particular limitation on the type of food. The food composition may be used in the form of formulations for oral administration such as tablets, hard or soft capsules, solutions, suspensions, etc. It can be prepared using a binder, disintegrant, lubricant, solubilizer, suspending agent, preservative or bulking agent. Examples of food include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, etc. Nutrients, etc. may be mentioned, but is not limited to these types of food.

The composition may be a food composition, preferably a beverage composition. The beverage composition of the present invention may be an alcoholic beverage. The alcoholic beverage means a beverage containing alcohol, more preferably an alcoholic beverage having a beer flavor prepared using hops or malt. For example, the alcoholic beverage may be sparkling wine. The alcoholic beverage of the present invention contains 0.1 to 8 v/v% of alcohol, preferably 0.5 to 7 v/v%, more preferably 1 to 6 v/v%, still more preferably 3 to 5.5 v / v% may be included. Also, the alcoholic beverage may be lager beer. However, the alcoholic beverage of the present invention is not limited thereto, and a beverage in which alcohol is removed after alcohol is produced through yeast fermentation is also included in the alcoholic beverage of the present invention.

The beverage may have a higher isoamyl acetate content than a beverage prepared by adding the parent strain, that is, wild-type Saccharomyces pastorianus.

Beverage manufacturing method

The present invention comprises the steps of adding Saccharomyces pastorianus HY2 to wort; And to a method for producing a beverage comprising the step of fermenting the wort to which the Saccharomyces pastorianus HY2 is added. In addition, the present invention relates to a method for producing a beverage comprising the step of adding and fermenting Saccharomyces pastorianus HY2.

At this time, in the method for producing a beverage of the present invention, in the method for producing beer, low-malt beer, etc., Saccharomyces pastorianus HY2 of the present invention may be used instead of the existing yeast in the conventional method for producing beer, low-malt beer, etc. .

The beverage may have a higher isoamyl acetate content than a beverage prepared by adding the parent strain, that is, wild-type Saccharomyces pastorianus.

Advantages and features of the present invention, and how to achieve them, will become clear with reference to the detailed description of the following embodiments. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various forms different from each other, only these embodiments make the disclosure of the present invention complete, and common knowledge in the art to which the present invention pertains. It is provided to completely inform the person who has the scope of the invention, and the present invention is only defined by the scope of the claims.

<Materials and methods>

The mother strain, wild-type Saccharomyces pastorianus, is a yeast suitable for lager beer production owned by Hite Jinro Co., Ltd. and was used in the present invention.

In addition, as a comparative group, S-189, a commercial lager Saccharomyces pastorianus strain from Fermentis, which is used for lager beer production, was purchased and used.

For the wort, the wort used in beer production by Hite Jinro Co., Ltd. was used.

<Experimental Example 1>

The isoamyl acetate content of domestic and foreign major lager beers (Test Groups A to F) was analyzed. As a result, the content of isoamyl acetate for each product varied from 0.84 to 2.66 ppm, and the banana flavor was not detected or weak during sensory evaluation (Table 1).

A B C D E F Isoamyl Acetate (ppm) 0.84 2.66 1.62 0.95 1.86 2.17

Results of isoamyl acetate content analysis of domestic and foreign beers (A to F)

<Experimental Example 2>

<Experimental Example 2-1> Testing

<2-1-1> EMS treatment condition test

EMS (Ethyl methanesulfonate) is a substance that induces random mutation by nucleotide substitution and was used as a mutagen for strain improvement.

Cells were recovered by centrifuging 1ml of the mother strain culture solution at 3,000 rpm for 5 minutes, and then washed three times with 0.85 w/v% sterile physiological saline solution. The treatment was hourly (20 to 150 minutes) in a shaking incubator at °C. Then, the strain suspension was centrifuged at 3,000 rpm for 5 minutes, the supernatant was discarded, washed once with an equal amount of 5 w/v% sodium thiosulfate solution and twice with 0.85 w/v% sterile physiological saline, and the viability of the strain was measured. did

<2-1-2> Selection of TFL-resistant strains (primary screening)

TFL (5,5,5-Trifluoro leucine) is a derivative structurally similar to leucine, and suppresses the synthesis of leucine by inhibiting α-IPM synthase in the isoamyl acetate biosynthetic pathway of the strain (FIG. 2). In addition, it is used for protein synthesis in competition with leucine, causing metabolic abnormalities to prevent the growth of strains. When the inhibitory mechanism of leucine feedback on α-IPM synthase is released, strains resistant to TFL can be selected and high production of isoamyl acetate is possible.

A minimal medium (2% glucose, 0.67% yeast nitrogen base w/o amino acids) containing TFL (100 ug/ml) was prepared and used as a selection medium for selecting strains with excellent ability to produce flavor.

The mother strain culture medium (1×10 ⁸ cells/ml) was repeatedly washed three times with 0.85 v/v% sterile physiological saline, and strain improvement was performed through EMS treatment. Thereafter, 100 μl of the strain suspension was spread on a TFL-containing selection medium and cultured at 25° C. for more than 4 days, and colonies with good proliferation were first selected.

<2-1-3> 500ml scale fermentation test (secondary screening)

In order to evaluate fermentation characteristics such as alcohol production and fragrance production ability, fermentation tests were performed by selecting 10 strains with excellent growth in a TFL-containing selective medium. At this time, a 500ml fermentation test was performed on 10 strains selected for the first time. After pitching at a concentration of 11 × 10 ⁶ cells / ml and fermenting at 14.5 ° C, the alcohol production ability was compared, and the content of each fragrance component was analyzed by Gas chromatography. was selected for the second time.

<2-1-4> 20L Pilot Fermentation Test - Isoamyl Acetate (Banana Flavor) High-producing strain selection

One) Comparison of fermentation characteristics

The mother strain, commercially available commercial strain (Fermentis S-189), and 4 secondary selected improved strains were pitched to 20L of wort at a concentration of 11×10 ⁶ cells/ml and fermented at 14.5° C. for 240 hours, Through fermentation characteristics (fermentation power) and aroma gas chromatography analysis, we tried to secure a strain with similar fermentation power to the parent strain and excellent fragrance production ability.

2) Prototype manufacturing and sensory evaluation

After completion of 20L fermentation, filtration was performed, and after diluting the fermented stock solution to an alcohol content of 4.5 v/v%, carbonation was performed to manufacture a prototype, and sensory evaluation was conducted on a research panel.

<Experimental Example 2-2> Test results

<2-2-1> EMS treatment condition test

As the time of treating the parent strain with EMS increased, the strain survival rate rapidly decreased and completely died after 120 minutes. As the EMS treatment time increases, the survival rate of the strain decreases and it is important to treat the EMS solution for an appropriate time because excessive genetic modification may cause unwanted changes. In order to obtain individuals with excellent traits, it is recommended to treat the EMS solution to have a survival rate of 50 to 60% (Winston, Molecular Biology, 2008), and in this test, the parent strain was suspended in a 5 v / v% EMS solution After treatment for 20 minutes, the survival rate was 64.4%. Therefore, it was determined that strain improvement was at an appropriate level, and the EMS treatment time for strain improvement was set to 20 minutes (FIG. 3).

<2-2-2> Selection of TFL-resistant strains (primary screening)

The concentration of the cells recovered after EMS treatment for 20 minutes in the parent strain was measured as 6.2×10 ⁷ cells/ml, and the survival rate was 64.4%. In order to select strains with excellent flavor production ability, a selection medium containing TFL was prepared, and 100 ul of the EMS untreated strain suspension and the EMS treated strain suspension were spread on the selection medium, respectively, and cultured at 25 ° C. for 4 days. When the EMS untreated strain suspension was spread on a TFL-containing selection medium, the growth of the cells could not be confirmed, but the EMS-treated strain suspension was able to confirm the growth of the strain on the TFL-containing selection medium. Among them, 10 strains with excellent growth were first selected (Table 2, Figure 4).

Control Trial TFL selective medium smear concentration
(before culture) 1.1x10 ^7cells /ml 6.2x10 ^6cells /ml TFL selection medium viable count (after culture) N.D. 3.3x10 ¹ cells/ml

Results of viable cell count measurement before and after cultivation in TFL selective medium

<2-2-3> 500ml scale fermentation test (secondary screening)

All 10 strains selected for the first time increased the production of isoamyl acetate by 28 to 103%, and in particular, strain HY2 showed the best isoamyl acetate production ability with an increase of 103% in isoamyl acetate production, inhibiting feedback by leucine. It was confirmed that did not occur (Table 3). In this case, the increase is based on the amount of isoamyl acetate produced by the parent strain, Control1. For example, a strain having an increased amount of 64% means that the amount of isoamyl acetate produced is 1.64 times that of Control1. Through this test, four strains, HY1, HY2, HY5, and HY8, which are judged to have excellent fragrance production ability because their fermentation ability is not inferior to the parent strain and are excellent in isoamyl acetate production, are selected and scaled up to 20L. A fermentation test was conducted. These four strains increased the isoamyl acetate production by about 75 to 103% compared to the parent strain in the 500ml scale fermentation test.

division strain name Isoamyl Acetate (ppm) increment
(%) Control1 mother strain 0.36 - Trial 1 HY1 0.63 75% Trial 2 HY2 0.73 103% Trial 3 HY3 0.59 64% Trial 4 HY4 0.64 78% Trial 5 HY5 0.65 81% Trial 6 HY6 0.56 56% Trial 7 HY7 0.46 28% Trial 8 HY8 0.65 81% Trial 9 HY9 0.54 50% Trial 10 HY10 0.55 53%

Evaluation of isoamyl production under 500 ml fermentation test conditions

<2-2-4> 20L Pilot fermentation test and sensory evaluation of the prototype

One) 20L Pilot Fermentation Test Results

Among the four strains selected in the 500ml fermentation test, a fermentation test with a capacity of 20 L was conducted to select a strain having no difference in fermentation characteristics from the parent strain and excellent isoamyl acetate producing ability. At this time, the parent strain was used as a control.

All four strains, HY1, HY2, HY5, and HY8, increased the amount of isoamyl acetate production by 106 to 164% compared to the parent strain, and in particular, strain HY2 showed the parent strain and fermentation characteristics (ie, alcohol production ability, fermentation power, final VDK concentration, yeast sedimentation, Recovery yeast dead cells, etc.) showed no significant difference, and there was no difference in alcohol production ability, and the production of isoamyl acetate increased by up to 164% compared to the parent strain, showing the best isoamyl acetate production ability, and feedback inhibition by leucine did not occur. It was confirmed that it was not (Table 4). Through this test, the fermentation characteristics were similar to the parent strain, and the HY2 strain, which showed the best flavor production ability, was secured, and the stability of fermentation was confirmed. There was no significant difference in fermentation characteristics of strain HY2 compared to the parent strain.

Control 1 Trial 1 Trial 2 Trial 5 Trial 8 mother strain HY1 HY2 HY5 HY8 Isoamyl Acetate (ppm) 1.40 3.10 3.69 2.89 3.12 Increase (%) - 121% 164% 106% 123%

Comparison of isoamyl acetate producing ability between parent strain and improved strain

In addition, the content of isoamyl acetate produced through a fermentation test with Saccharomyces pastorianus HY2 strain obtained through the present invention and S-189, a commercial lager strain from Fermentis, which is widely used in lager beer production, was compared.

As a result, it was found that the isoamyl acetate content increased by about 77% when fermented with Saccharomyces pastorianus HY2, an improved strain of the present invention, compared to Fermentis' commercial lager strain, S-189 (Table 5). At this time, the amount of isoamyl acetate increase is calculated based on the amount of isoamyl acetate produced by Control 2, S-189.

Control 2 Trial 2 Fermentis
S-189 HY2 Isoamyl Acetate (ppm) 2.09 3.69 Increase (%) - 77%

Comparison of isoamyl acetate producing ability between strain S-189 and strain HY2 for lager beer production

One) 20L Pilot prototype sensory evaluation

In order to compare the beer taste sensations of the mother strain obtained in the above test, the commercial strain S-189 from Fermentis, and the HY2 strain, after diluting the alcohol content of the fermentation stock solution to 4.5 v / v%, 10 laboratory panels A characteristic difference test (5-point scale) was conducted on the subjects. 1 point: Very weak, 3 points: Normal, 5 points: Very strong.

As a result, it was found that the banana flavor of the HY2 strain was strong and the flavor harmony was excellent compared to the parent strain and the commercial strain S-189. In addition, the preference for the HY2 strain was also higher than that of the parent strain and commercial strain S-189. Therefore, it was confirmed that beer having excellent banana flavor characteristics due to isoamyl acetate could be prepared using the Saccharomyces pastorianus HY2 strain (Table 6).

Accordingly, the Saccharomyces pastorianus HY2 strain was deposited (accession number: KFCC11896P). Saccharomyces pastorianus HY2 of the present invention is a lager yeast having an excellent ability to produce isoamyl acetate imparting a banana flavor, and it was judged to be useful in preparing a banana flavor-enhanced beverage, preferably beer or sparkling wine.

flavor
degree of harmony banana flavor body refreshing feeling Sour taste preference mother strain 3.0 3.0 3.0 3.1 1.5 3.5 Commercial strain S-189 3.0 2.8 3.1 3.1 1.5 3.4 Improved strain HY2 3.8 4.3 3.3 3.3 1.5 3.9

Comparative results of sensory evaluation of prototypes using parent strain, commercial strain and improved strain

<Synthesis of test results>

go. By applying the EMS strain improvement method, a strain selection test with excellent isoamyl acetate (banana flavor) production ability was conducted. It is recommended. In this test, after suspending the parent strain in 5 v / v% EMS solution, when treated for 20 minutes, the survival rate was 64.4%, so the EMS treatment time for strain improvement was set to 20 minutes.

me. In a selective medium containing TFL, strains that do not have resistance to TFL do not grow, and strains in which the feedback inhibition mechanism by leucine is released through EMS treatment are resistant to TFL and can grow in a selective medium containing TFL. Through this test, 33 colonies were identified in the TFL-containing selection medium, and among them, 10 strains with good growth were first selected.

all. As a result of the 500ml fermentation test on the 10 strains selected for the first time, all 10 strains increased the production of isoamyl acetate by 28 to 103% compared to the parent strain, and in particular, the HY2 strain increased the production of isoamyl acetate by 103%, producing the best isoamyl acetate. showed ability. Through this test, four strains of HY 1, 2, 5, and 8 with similar fermentation ability to the parent strain and excellent isoamyl acetate production were selected.

la. As a result of the 20L fermentation test, it was confirmed that the HY2 strain had similar fermentation characteristics to the parent strain, and that the production of isoamyl acetate increased by 164% compared to the parent strain. In addition, compared to the S-189 strain, which is a commercial lager strain that is widely used worldwide, it was confirmed that the HY2 strain increased the amount of isoamyl acetate production by 77% compared to the S-189 strain. Through this test, the fermentation characteristics were similar to the parent strain and the HY2 strain, which showed the best flavor production ability, was selected as the yeast with the best isoamyl acetate (banana flavor) production ability.

mind. After diluting the alcohol content to 4.5 v / v% with the fermentation broth obtained in the fermentation test, a prototype was prepared, and a sensory characteristic difference test was performed on the laboratory panel. 189 prototype, and preference was also high.

bar. In order to select strains with excellent ability to produce isoamyl acetate (banana flavor), a strain improvement process by EMS treatment was established, and through the present invention, one HY2 strain with excellent ability to produce isoamyl acetate (banana flavor) was secured. Therefore, it was determined that the use of the Saccharomyces pastorianus HY2 strain could further improve the flavor of beer and produce a unique beer.

Name of Depositary Institution: Korea Center for Microbial Conservation (Korea)

Accession number: KFCC11896P

Entrusted date: 20210908

Claims (8)

Saccharomyces pastorianus HY2 (accession number KFCC11896P).
According to claim 1,
Saccharomyces Pastorianus HY2 is Saccharomyces Pastorianus HY2, characterized in that it has better isoamyl acetate producing ability than wild-type Saccharomyces Pastorianus.
A composition comprising Saccharomyces pastorianus HY2 of claim 1, a culture thereof, a concentrate of said culture, or a dilution of said culture.
According to claim 2,
The composition is a composition, characterized in that the food composition.
According to claim 2,
The composition is a composition, characterized in that the beverage composition.
Adding Saccharomyces pastorianus HY2 of claim 1 to wort; and
A method for producing a beverage comprising the step of fermenting the wort to which the Saccharomyces pastorianus HY2 is added.
According to claim 6,
The method of claim 1, wherein the beverage has a higher isoamyl acetate content than a beverage prepared by adding wild-type Saccharomyces pastorianus.
A method for producing a beverage comprising the step of adding and fermenting Saccharomyces pastorianus HY2 of claim 1.

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